Revisiting NaV Channel Inactivation: The Role of Fibroblast Growth Factor Homologous Factor (FHF)

Abstract

Cardiac sodium channel (NaV1.5) activation underlies the initiation and propagation of the heartbeat and is regulated by numerous accessory proteins. NaV1.5 comprises 4 homologous domains (DI-DIV), each consisting of 6 transmembrane segments (S1-S6) constituting the voltage sensing domain (VSD; S1-S4) and the channel pore (S5-S6). FHF, or intracellular fibroblast growth factors (iFGF), were found to regulate NaV1.5 kinetics through C-terminal binding near Calmodulin (CaM), but the mechanism remains unclear. To probe this mechanism, we observed the regulation of Nav1.5 VSDs by the cardiac-predominant FHF1B (iFGF12B), and the alternative FHF1A (iFGF12A) splice variant, using voltage-clamp fluorometry (VCF) in the cut-open configuration for individual VSD monitoring. Voltage clamp and patch clamp were also employed for electrophysiological studies in Xenopus Oocytes and CHO cells. We found that FHF1A and FHF1B regulate NaV1.5 inactivation by causing depolarizing shifts in the steady-state inactivation (SSI) curve and the DIII-VSD activation. Disruption of the CaM binding motif (IQ/AA) did not alter the SSI shift, suggesting CaM-independent FHF modulation. However, mutation of the inactivation gate (IFM motif) within DIII-IV linker (F1486Q) negates the FHF's effects on DIII-VSD and SSI. We conclude that FHF1s regulate NaV1.5 inactivation through an inactivation gate, independent of CaM. Furthermore, the A-type FHF counteracts impaired inactivation of F1486Q-NaV1.5 and reduces the late sodium current. A peptide bearing the first 21 amino acids of FHF1A's N-terminus is insufficient to reduce the late sodium current, implying different mechanism than pore blocking. Similar inactivation rescue by FHF1A was observed in long QT syndrome-linked mutations (ΔKPQ, ΔK1500 and F1473S), without affecting the shifts in SSI nor DIII-VSD. Results suggest possible FHF1A function as natural antiarrhythmic agent, that partially restore the inactivation impaired by mutations through an inactivation gate stabilization, independent of its connection to DIII-VSD.